Diagnostic assays are used in a variety of contexts for sample analysis. The assay may be for detecting the presence of specific analytes or used to assess the structural integrity or morphological changes in the sample being analyzed. For example, in the clinical laboratory, immuno-based assays are used to detect a myriad of analytes diagnostic of particular disease conditions. These assays may detect the presence of pathogenic organisms, such as viruses and bacteria; identify levels of a specified compound indicative of a disease condition; or reveal markers for cells and tissues involved in the disease process. In the area of analytical chemistry, analytical assays provide a rapid and simple method for detecting various organic and inorganic compounds, particularly for initial tests of a sample or as an adjunct to highly sensitive procedures such GC/mass spectroscopy and atomic absorption spectroscopy. For instance, the presence of antimony, barium, and lead found in firearm discharges are readily determined by reaction with sodium rhodizonate, which forms a colored product with the metals. Similarly, nitrates present as ammonium nitrate in explosives react with diphenylamine or diphenylamine derivatives to generate visible products.
In part, the sensitivity and the reproducibility of any such assays are affected by the quality of the assay reagents. Purity of ingredients used to prepare the reagents can vary. In addition, certain reagents degrade over time or are unstable under various physical conditions, such as temperature, pH, and light. Reagents also react with other reagents or the solvent, thus altering the reactivity and availability of the reagent. Since many standard clinical diagnostic assays are sold commercially in kit form, there will be batch-to-batch differences in the reagents because of manufacturing variations, even when commercial suppliers institute GMP (good manufacturing practices) standards.
Moreover, laboratory-to-laboratory performance of the assay can vary. This may arise from different operating procedures used in laboratories in terms of storage and handling of reagents. Additionally, the technician's skill, experience, and training can affect the quality of the assay result.
In order to generate consistency and accuracy in any diagnostic assay, it is beneficial to have some sort of quality assurance to validate the assay and the results obtained. This generates confidence in the data, and points out any problems that may arise in performing the assay. Validation of assay performance becomes critical with increasing complexity of diagnostic procedures, particularly where the assay involves a multitude of reagents and multiple process steps. For instance, an immunohistochemistry based diagnostic procedure practiced in a clinical laboratory may use an indirect conjugate or sandwich technique to determine the presence of a target analyte. Typically, this assay format involves exposure of hydrated slides containing a tissue sample to a primary antibody, which has no modifications to the antibody itself. This step is followed by exposure to a secondary antibody directed against the species in which the first antibody was raised. The secondary antibodies are typically composed of a mixture of antibodies (i.e., polyvalent), and may be obtained from a variety of animal species commonly used in the art to generate the primary antibody. Secondary antibodies have modifications that are capable of generating a visible staining reaction at sites where the primary antibody is bound to the specimen. To increase the detectable signal, secondary antibodies are commonly conjugated to small molecule ligands, such as biotin, capable of binding with high affinity to a cognate binding partner. After the secondary antibody step, the specimens are reacted with the high affinity binding partner, which typically has a label, such as an enzyme that acts on a suitable substrate (i.e., chromogen), to generate a visible, colored product in subsequent staining steps.
As described, this sandwich type immunostaining protocol has several points where amplification occurs: (1) at binding of the secondary antibody to the primary antibody, (2) at binding of the small molecule ligands to the high affinity molecule, and (3) at the enzyme action on the chromogenic substrate. The level of amplification at each of these points is difficult to evaluate because, typically, only the final signal, the presence of the colored product, is generally determined. Thus, it is difficult and time consuming to identify variations in reagent quality at each step of the assay and whether each step is working optimally. Moreover, due to the complex number of steps involved in the staining protocol, technical mistakes (e.g., omissions of steps) can be common, resulting in failures of the staining protocol.
Use of a known positive specimen does provide some level of control for assessing the staining procedure, but suffers from the problem that most methods of specimen fixation and processing affect the final signal obtained. Thus the actual stain intensity achieved on the control specimen compared to the unknown specimen cannot be compared in any quantitative fashion.
Thus it would be highly desirable to provide a way to verify that an assay protocol having multiple reagents and multiple process steps has been performed properly, as well as an assessment of the potential changes in reagent quality over time, and that an appropriate result was obtained.